The present invention provides sensors to monitor protein kinase activity continuously with a fluorescence readout. The sensor requires minimal perturbation of a protein kinase peptide substrate. The fluorescence response with respect to time over the course of the reaction corresponds to enzyme activity. The sensor of the present invention can be used in high-throughput screening of inhibitors or substrates, detection of activity in cell extracts or enzyme purifications, spatial or temporal localization of kinase activity in a cell, and elucidation of complicated signal transduction pathways.
Protein kinases are involved in all aspects of regulation within cells. A protein kinase catalyzes the transfer of a phosphate group from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in a peptide or protein sequence. Each kinase is specific for the amino acids surrounding the residue to be phosphorylated. The traditional method for assaying kinase activity is discontinuous and requires 32P-labelled ATP, which requires special handling. Many companies market specialized fluorescence kinase assay systems, all of which are discontinuous, requiring sampling of the reaction mixture followed by additional handling steps to label the product of the reaction with a fluorescent moiety (e.g., Promega, Panvera, Calbiochem, Cell Signaling Technology, Molecular Devices, DiscoveRx, Upstate, PerkinElmer). A continuous fluorescence assay that can be performed in real time is of great utility. Currently, few examples of sensors capable of such assays exist.
Approaches include: environment-sensitive fluorophores near the phosphorylation site (Wright, D. E. et al. Proc. Natl. Acad. Sci. USA 1981, 78, 6048-6050; McIlroy, B. K. et al. Anal. Biochem. 1991, 195, 148-152; Higashi, H. et al. FEBS Lett. 1997, 414, 55-60; Post, P. I. et al. J. Biol. Chem. 1994, 269, 12880-12887), FRET pairs flanking a sequence which undergoes a conformational change upon phosphorylation (Nagai, Y. et al. Nat. Biotech. 2000, 18, 313-316; Ohuchi, Y. et al. Analyst 2000, 125, 1905-1907; Zhang, J. et al. Proc. Natl. Acad. Sci. USA 2001, 98, 14997-15002; Ting, A. Y. et al. Proc. Natl. Acad. Sci. USA 2001, 98, 15003-15008; Hofmann, R. M. et al. Bioorg. Med. Chem. Lett. 2001, 11, 3091-3094; Kurokawa, K. at el. J. Biol. Chem. 2001, 276, 31305-31310; Sato, M. et al. Nat. Biotech. 2002, 20, 287-294; Violin, J. D. et al. J. Cell Biol. 2003, 161, 899-909), or Ca2+ chelation between the phosphate and in internal chelator causing disruption of PET-quenching (Chen, C.-A.; et al. J. Am. Chem. Soc. 2002, 124, 3840-3841). A majority of these sensors have very modest fluorescence increases or sometimes decreases, with the notable exception of 1.5-2.5-fold increases in the probes reported by Lawrence and coworkers (Chen 2002, supra; Yeh, R.-H.; et al. J. Biol. Chem. 2002, 277, 11527-11532). However, these types of probes, with fluorophores adjacent to the phosphorylated residue or very large fluorophores may interfere with their recognition by and reactivity with certain kinases.